Lactococcus lactis NZ9010 in which the las operon-encoded ldh gene was replaced with an erythromycin resistance gene cassette displayed a stable phenotype when grown under aerobic conditions, and its main end products of fermentation under these conditions were acetate and acetoin. However, under anaerobic conditions, the growth of these cells was strongly retarded while the main end products of fermentation were acetate and ethanol. Upon prolonged subculturing of this strain under anaerobic conditions, both the growth rate and the ability to produce lactate were recovered after a variable number of generations. This recovery was shown to be due to the transcriptional activation of a silent ldhB gene coding for an Ldh protein (LdhB) with kinetic parameters different from those of the native las operon-encoded Ldh protein. Nevertheless, cells producing LdhB produced mainly lactate as the end product of fermentation. The mechanism underlying the ldhB gene activation was primarily studied in a single-colony isolate of the recovered culture, designated L. lactis NZ9015. Integration of IS981 in the upstream region of ldhB was responsible for transcription activation of the ldhB gene by generating an IS981-derived ؊35 promoter region at the correct spacing with a natively present ؊10 region. Subsequently, analysis of 10 independently isolated lactate-producing derivatives of L. lactis NZ9010 confirmed that the ldhB gene is transcribed in all of them. Moreover, characterization of the upstream region of the ldhB gene in these derivatives indicated that site-specific and directional IS981 insertion represents the predominant mechanism of the observed recovery of the ability to produce lactate.Homolactic fermentation by lactic acid bacteria involves the classical Embden-Meyerhoff-Parnas pathway leading to pyruvate, which is converted to lactic acid by lactate dehydrogenase. This enzyme and the gene that encodes it have been studied in many lactic acid bacteria, including Lactococcus lactis (11, 34), Streptococcus thermophilus (19), and various lactobacilli (2,15,47,51). L. lactis is the best-studied representative of this group, and the complete and partial genomes of several strains have been determined (4, 29). The gene encoding L. lactis Ldh was identified and characterized by Llanos and coworkers in 1992 (33, 34). The ldh gene is the last gene of the so-called lactic acid synthesis or las operon, which also encodes the glycolytic enzymes phosphofructokinase and pyruvate kinase. Transcription of the las operon was shown to yield a polycistronic transcript encompassing all three genes. But under some conditions, transcripts representing only two genes (pfk and pyk or pyk and ldh) or even a single gene (ldh) of the operon were also detected, which probably resulted from RNA processing upstream of the pyk and ldh genes (38). It has been shown that the las operon is subject to CcpAmediated carbon catabolite transcriptional activation, and a CcpA target site (cre sequence) was found within the las promoter region (38). The...